Methods for nucleic acid amplification and detection of amplification products may be used to detect, identify, quantify and analyze nucleic acid sequences. Nucleic acid amplification is an important step in the construction of libraries from related genes such as antibodies.
These libraries can be screened for antibodies having specific desirable activities. Nucleic acid analysis is important for a variety of purposes such as, for example, detection and identification of pathogens, detection of gene alteration leading to defined phenotypes, diagnosis of genetic diseases or the susceptibility to a disease, assessment of gene expression in development, and in response to defined stimuli, as well as the various genome projects. Other applications of nucleic acid amplification methods include the detection of rare cells, detection of pathogens, detection of altered gene expression in malignancy, etc.
Amplification methods that employ a single primer are disclosed in U.S. Pat. Nos. 5,508,178; 5,595,891; 5,683,879; 5,130,238; and 5,679,512. In U.S. Pat. No. 5,744,308, the primer is a DNA/RNA chimeric primer. Alternative amplification methods that employ template switching oligonucleotides (TSOs) and blocking oligonucleotides are disclosed in U.S. Pat. Nos. 5,679,512; 5,962,272; and 6,251,639. In some of these amplification methods, the TSO amplification method utilizes chimeric DNA primer.
Nucleic acid amplification is also useful for qualitative analysis (such as, for example, the detection of the presence of defined nucleic acid sequences) and quantification of defined gene sequences (useful, for example, in assessment of the amount of pathogenic sequences as well as the determination of gene multiplication or deletion and cell transformation from normal to malignant cell type). The detection of sequence alterations in a nucleic acid sequence is important for the detection of mutant genotypes, as relevant for genetic analysis, the detection of mutations leading to drug resistance, pharmacogenomics, etc.
There are many variations of nucleic acid amplification: for example, exponential amplification, linked linear amplification, ligation-based amplification, and transcription-based amplification. One example of exponential nucleic acid amplification method is polymerase chain reaction (PCR), which has been disclosed in numerous publications. Indeed, PCR is the most commonly used target amplification method. PCR is based on multiple cycles of denaturation, hybridization of two different oligonucleotide primers, each to opposite strand of the target strands, and primer extension by a nucleotide polymerase to produce multiple double stranded copies of the target sequence.
Traditional DNA amplification requires two gene-specific primers for PCR. As each primer has its own optimal annealing temperature, it is not easy to amplify a particular gene specifically and with good efficiency. This is especially a concern for amplification of antibody genes. In these methods, the forward primers tend to anneal to the framework 1 region of the antibody genes where some somatic mutations and variations may necessitate the use of lower annealing temperatures to allow some ambiguities. However, this is not optimum for the reverse primers where they usually anneal to the constant region of the antibody genes and leads to non-specific amplification of other genes. These factors considerably lower the specificity of the amplified genes and covered repertoires after the amplification and result in an overall skewed diversity and loss of rare but important genes.